The ability to rapidly characterize molecular function and/or activation of oncogenes in living cells would be a fundamental advance in the diagnosis of cancer, the planning of molecular-based therapies, and in cancer prevention. Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer that is highly angiogenic, invasive, and metastatic. RhoC-GTPase has been identified in the Merajver lab as a specific marker of aggressive breast cancer phenotypes and of IBC in particular. RhoC behaves as a transforming oncogene of human mammary epithelial cells whose overexpression can lead to a highly invasive, angiogenic, and metastatic phenotype, extremely akin to IBC. The biophysical mechanisms for activation and inhibition of this oncogene (including detailed molecular associations and cellular localization) are not completely understood; however, these features impact on function and biological activity in tumors. We propose in this R21 application to investigate RhoC-GTPase localization, activation, and inhibition in living human cellular models of IBC using intensity and lifetime fluorescence resonance energy transfer (FRET) technology and methods developed in the Mycek lab for cellular and molecular imaging. FRET measurements detect molecular localization and binding with nanoscale spatial sensitivity. The use of fluorescence lifetime sensing offers an additional source of contrast for performing quantitative FRET measurements in living cells, while being generally independent of artifacts influencing fluorescence intensity (including fluorophore concentration, photobleaching, and sources of optical loss (absorption and scattering) in biological systems) that may complicate the interpretation of intensity measurements. Specific Aim 1: Characterize RhoC-GTPase oncogene localization in living normal mammary epithelial cells and in RhoC-driven malignant cells. Specific Aim 2: Determine RhoC activation states with and without binding to the key effector, rhotekin. Specific Aim 3: Define the changes in FRET and RhoC localization caused by known inhibitors of RhoC-GTPase activation: C3 exotransferase and a farnesyl transferase inhibitor (FTI 832). The molecular imaging methods developed in this R21 proposal are cross-cutting in nature in that they are broadly applicable to basic biological studies of cellular processes and structures in living cells obtained from patients, and would find rapid translation to the clinic.